We examined parathyroid hormone-related peptide (PTHrP) production and regulation in both normal human melanocytes and in a human amelanotic melanoma cell line (A375). Northern blot and immunocytochemical analysis demonstrated that both cultured A375 cells and normal human melanocytes express PTHrP, but A375 cells expressed much higher levels of the peptide. PTHrP secretory rate increased at least 10-fold after treatment with 10% fetal bovine serum (100.2 ± 2.8 pmol/10⁶ cells vs. basal <15 pmol/10⁶ cells) in proliferating A375 cells but only twofold in confluent cells. Treatment of A375 cells with increasing concentrations of 1,25-dihydroxyvitamin D₃[1,25-(OH)₂D₃] or its low-calcemic analog EB-1089 revealed that EB-1089 was 10-fold more potent than 1,25-(OH)₂D₃ on inhibition of both cell proliferation and PTHrP expression. Furthermore, inoculation of A375 cells into the mammary fat pad of female severe combined immunodeficiency mice resulted in the development of hypercalcemia and elevated concentrations of plasma immunoreactive PTHrP in the absence of detectable skeletal metastases. Our study, therefore, demonstrates a stepwise increase in PTHrP expression when cells progress from normal to malignant phenotype and suggests that EB-1089 should be further evaluated as a therapeutic agent in human melanoma.